Campylobacter Polypeptides and Methods of Use

ABSTRACT

The present invention provides isolated metal regulated polypeptides obtainable from a  Campylobacter  spp., and compositions including the polypeptides. The present invention also includes methods for using the compositions disclosed herein, including methods for treating in infection in a subject, for treating a condition caused by a  Campylobacter  spp., and for decreasing colonization of an animal.

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Ser.No. 60/504,119, filed 19 Sep. 2003, which is incorporated by referenceherein.

BACKGROUND

Campylobacter spp. is part of the normal intestinal flora of a widerange of domestic and wild animals with a particular niche for the avianhost. Campylobacter spp. do appear to have a limited ability to bepathogenic in domestic and wild animals. In cattle C. fetus subsp.jejuni and C. fetus subsp. intestinalis have been isolated fromintestines and experimentally transmitted to preruminant and ruminantcalves which developed clinical signs of fever, diarrhea and sporadicdysentery (Dannenberg et al. Am. J. Pathol. 34: 1099 (1958) and Thomaset al. Aust. Vet. J. 36: 146 (1981)). A syndrome of profuse waterydiarrhea with fever, anorexia and depression in yearling sheep has alsobeen reported with Campylobacter fetus as the causative agent.Campylobacter spp. has also been reported to cause clinicalmanifestations of dysentery, intestinal adenomatosis and hemorrhagicenteritis in pigs and horses, and mastitis in commercial dairy herds.

In humans, Campylobacter is the most commonly reported bacterial causeof endemic diarrheal illness worldwide. In the United States it isbecoming the most prevalent cause of foodborne infection and affectsmore than 2 million people annually. In England and Wales, over 50,000campylobacter cases are reported annually with no signs of decline ofincidence. It is estimated that for every case reported to laboratorysurveillance, another seven cases occur unreported. C. jejuni and C.coli are the two most commonly isolated species responsible for humanCampylobacteriosis with C. jejuni now being the most frequentlyisolatable species.

The incubation period following ingestion of C. jejuni has been shown tobe approximately 24-72 hours. The inoculum size required to induceclinical symptoms has been shown to be as few as 800 organisms. The rateof illness increases with increasing numbers of the organism ingested.Commonly reported symptoms of human Campylobacteriosis include diarrhea,fever, and abdominal cramping. Less frequently, Campylobacter,particularly C. jejuni, can cause secondary sequelae following an acuteinfection, including, reactive arthritis, kidney failure,Guillian-Barre, Reiter syndrome and other extra-intestinal symptoms.

The transmission of Campylobacter spp. to human populations is primarilythrough environmental contamination and contaminated foods, includingpoultry and poultry products such as eggs. Campylobacter spp. can beisolated from 30-100% of the birds in many domestic and wild avianspecies at any given time. In children, contact with puppies and kittenswith diarrhea has been shown to be an important additional risk factor.Some additional sources of infection have resulted from drinking rawmilk derived from cows having clinical mastitis caused by Campylobacter.All milk-borne outbreaks have been associated with raw or improperlypasteurized milk.

The virulence and pathogenesis of Campylobacter spp. involves both hostand pathogen specific factors. Many pathogen-specific virulencedeterminants contribute to the pathogenesis of these bacteria. Thebacterial virulence of these bacteria is the result of many differentattributes, which often contribute to different steps in the complicatedseries of events recognized as an infection. Exposure first takes placeprimarily by the consumption of contaminated water, food or by directperson to person contact. Once ingested the stages of infection commonto these bacteria include attachment, colonization, proliferation,tissue damage, invasion and dissemination.

The first host barrier that Campylobacter must typically overcome is themucosal surface. A single epithelial cell layer separates the host fromthe lumen of the gastrointestinal tract. This barrier and a plethora ofother host antimicrobial mechanisms deter commensal, opportunistic andpathogenic microorganisms from establishing infection. Adherence tomucosal surfaces is a prerequisite of this pathogen to establishinfection. One of the more pronounced clinical manifestations ofintestinal colonization is diarrhea. This clinical syndrome has beenproposed to be produced by the synthesis and excretion of enterotoxinsthat cause a net secretion of fluid and electrolytes (diarrhea). Otherspecific virulence factors include flagella, which assist the bacteriumto overcome the clearing movement of peristalsis and enable the organismto enter and cross the mucous layer covering the epithelium (Black etal., J. Infect. Dis. 157:472-479 (1988), Caldwell et al., Infect Immun.50:941-943 (1985), Morooka et al., J. Gen. Micro. 131:1973-1980 (1980)and Newell et al. J. Hyg. Camb. 95:217-227 (1985)). Other suspecteddeterminants of pathogenicity include chemotaxis, iron-acquisition, hostcell invasion, inflammation and active secretion and epithelialdisruption with leakage of serosal fluid (Black et al. J. Infect. Dis.157: 472-479 (1988)).

Divalent metal ions such as iron, cobalt, copper, magnesium, manganese,molybdenum, nickel, selenium, and zinc are trace elements often requiredfor the survival of bacteria infecting both animal and human hosts.These trace metal elements are used by bacteria as cofactors for enzymesthat catalyze biochemical reactions for various metabolic pathways andtransport systems required by the organism. The metals iron, zinc andmanganese are the three most important metals required for the survivalof bacteria. Zinc ions are essential for RNA and DNA polymeraseactivity, whereas manganese is required for mitochondrial superoxidedismutase activity. Iron is the most extensively studied of all themetal ions with direct correlations on the virulence and pathogenesis ofbacteria. Iron is essential for all life and is required for enzymaticand metabolic pathways of organisms at all phylogenic levels.

The ability of Campylobacter to evade the natural defense mechanisms ofthe vertebrate host depends in part on its ability to obtain host iron,which in turn directly influences the host-pathogen interaction. Becauseof iron's essential nature, vertebrate hosts have developed elaboratemechanisms to bind iron in body fluids (e.g., transferrin in blood andlymph fluids and lactoferrin in external secretions). These highaffinity iron binding proteins create an iron restricted environmentwithin the host reducing the level of iron to approximately 10⁻¹⁸ molar,a concentration too low to support the growth of nearly all bacteria.These iron sequestering mechanisms of the host act as a natural defensemechanism to combat bacterial invasion. To circumvent theseiron-restrictive conditions many bacterial species have evolvedmechanisms for obtaining iron. The most common mechanisms include thediffusion of soluble iron through porins and specialized transportsystems that mediate the uptake of iron by siderophores. This lattersystem is by far the most widespread or ubiquitous mechanism for ironacquisition and involves the specific chelation of ferric iron bysiderophores and the synthesis of their cognate transport systems, whichpermits the bacteria to continue to replicate and overcome thenon-specific defense mechanisms of the host. Continued replication, andthus each step in the infectious process, is ultimately dependent on theability of the organism to obtain iron from its host.

With so many basic functions relying on the availability of iron,bacteria have evolved a complex regulatory network for acquiring ironunder varying physiological conditions. Iron is a divalent cation whichexists both in the ferrous (Fe²⁺) state and in the ferric (Fe³⁺) state.Under anaerobic conditions, iron is present in the soluble ferrous form(Fe²⁺) and can freely diffuse through outer membrane porins into theperiplasm. For instance, in E. coli the FeoAB transport system presentin the cytoplasmic membrane will transport the ferrous iron moleculesinto the cell cytoplasm. Under aerobic conditions and neutral pH, ironis primarily present in the insoluble ferric form (Fe³⁺) and cannot passthrough the outer membrane porins by passive diffusion. Instead,molecules called siderophores are secreted by bacteria, which have ahigh affinity for ferric iron. The ferric-siderophore complexes arerecognized by receptors in the outer membrane, collectively referred toas the TonB-dependent receptors. These receptors, once bound to loadedsiderophores, are believed to interact with TonB and its associatedproteins localized in the periplasm and cytoplasmic membrane. Theseprotein-protein interactions, though poorly understood, serve to providethe energy necessary to transport the ferri-siderophore complexes acrossthe outer membrane and through the periplasmic space. ABC transportsystems present in the cytoplasmic membrane serve to transport theiron-siderophore complexes across the cytoplasmic membrane. Reductaseenzymes then serve to reduce ferric iron to its ferrous form, whichdissociates it from the siderophore and releases iron into the cell.

Several species of pathogenic bacteria use additional mechanisms toobtain iron from mammalian hosts, including the direct binding of hemeand hemoglobin. The receptor proteins that bind these iron-containingmolecules most likely rely on the TonB complex for the energy requiredto transport heme across the outer membrane, similar to theiron-siderophore complexes. Specialized ABC transporters are then usedto transport the heme across the cytoplasmic membrane. In addition, somebacteria secrete hemophores, small molecules that can bind heme andpresent it to receptors on the bacterial cell surface. Severalpathogenic species also produce hemolysins, which are toxins that lysered blood cells, releasing heme and hemoglobin for uptake by thebacteria.

The outer membrane proteins of gram-negative bacteria control theselective permeability of many essential nutrients critical to thesurvival of bacteria, including all pathogenic bacteria that causedisease in animals and man. This selective permeability of nutrients iscontrolled by a class of membrane proteins called porins. It now appearsthat the majority of the outer membrane proteins on the surface ofgram-negative bacteria are porins, identified as the general porins(e.g., OmpF), monomeric porins (e.g., OmpA), the specific porins (e.g.,the maltose-specific porin LamB) and the TonB-dependent, gated porins(e.g., the siderophore receptor FepA). The porin class of proteinsgenerally share structural features, including the presence ofbeta-barrels that span the outer membrane.

Little is known regarding the iron-acquisition by Campylobacter spp.Studies indicate that C. jejuni does not synthesize siderophores (Fieldet al. Infect. Immun. 54: 126-132 (1986) and Picket et al. Infect Immun.60: 3872-3877 (1992)). This data has been confirmed by sequence analysisof C. jejuni genome in which no homologs of common siderophore synthesisgenes were identified. C. jejuni is limited in the iron compounds it canuse as demonstrated by various feeding assays. These assays havedemonstrated that C. jejuni can use the siderophores enterochelin andferrichrome but not aerobactin, desferal, ferritin, lactoferrin, ortransferrin. Therefore, it has been suggested that other iron compoundsare required to support the growth of Campylobacter spp. such as hemecompounds like hemin and hemoglobin, ferric iron, and ferrous iron. Thefact that Campylobacter has known transport systems for siderophores,yet is unable to synthesize them, suggests that these bacteria scavengesiderophores produced by other enteric pathogens (Arnoud et al. FEMSMicrobiol. Rev. 26: 173-186 (2002)).

SUMMARY

The present invention provides an isolated metal regulated polypeptideobtainable from a Campylobacter spp., wherein the polypeptide isexpressed by a Campylobacter spp. at a detectable level during growthunder low metal conditions and is not expressed by the Campylobacterspp. at a detectable level during growth in high metal conditions, and acomposition including the polypeptide. The isolated metal regulatedpolypeptide may have a molecular weight of between 150 kDa and 152 kDa,between 143 kDa and 145 kDa, between 123 kDa and 125 kDa, between 92 kDaand 94 kDa, between 88 kDa and 90 kDa, between 73 kDa and 75 kDa,between 69 kDa and 71 kDa, between 51 kDa and 53 kDa, between 50 kDa and52 kDa, or between 38 kDa and 40 kDa. The composition may furtherinclude a second metal regulated polypeptide having a molecular weightof between 57 kDa and 59 kDa, between 54 kDa and 56 kDa, between 47 kDaand 49 kDa, between 42 kDa and 44 kDa, between 37 kDa and 39 kDa, orbetween 28 kDa and 30 kDa, wherein the second polypeptide is expressedby a Campylobacter spp. during growth in high metal conditions andexpressed at an enhanced level during growth in low metal conditions.

The present invention also provides an isolated metal regulatedpolypeptide obtainable from a Campylobacter spp., wherein thepolypeptide is expressed by a Campylobacter spp. during growth in highmetal conditions and expressed at an enhanced level during growth in lowmetal conditions, and a composition including the polypeptide. The metalregulated polypeptide may have a molecular weight of between 57 kDa and59 kDa, between 54 kDa and 56 kDa, between 47 kDa and 49 kDa, between 42kDa and 44 kDa, between 37 kDa and 39 kDa, or between 28 kDa and 30 kDa.

The composition may further include a second metal regulated polypeptidehaving a molecular weight of between 150 kDa and 152 kDa, between 143kDa and 145 kDa, between 123 kDa and 125 kDa, between 92 kDa and 94 kDa,between 88 kDa and 90 kDa, between 73 kDa and 75 kDa, between 69 kDa and71 kDa, between 51 kDa and 53 kDa, between 50 kDa and 52 kDa, or between38 kDa and 40 kDa, wherein the second polypeptide is expressed by aCampylobacter spp. at a detectable level during growth under low metalconditions and is not expressed by the Campylobacter spp. at adetectable level during growth in high metal conditions.

The present invention also includes methods for using the compositionsdisclosed herein, including methods for treating in infection in asubject, for treating a condition caused by a Campylobacter spp., fordecreasing colonization of an animal. The methods include administeringan effective amount of a composition to an animal, where the compositionincludes an isolated metal regulated polypeptide obtainable from aCampylobacter spp.

Also included in the present invention is a composition including anisolated whole cell preparation of a Campylobacter spp., wherein thecells include either a metal regulated polypeptide expressed by theCampylobacter spp. during growth under low metal conditions and notexpressed during growth in high metal conditions, a metal regulatedpolypeptide expressed by the Campylobacter spp. during growth in highmetal conditions and expressed at an enhanced level during growth in lowmetal conditions, or the combination thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Gel-image of Campylobacter jejuni extracted membrane proteinprofile expressed under iron-replete and iron-deplete growth conditions.

FIG. 2. The difference in fecal shedding between vaccinated andnon-vaccinated mice after oral challenge with Campylobacter jejuni. Log10 CFU, mean number of bacteria in fecal sample.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides polypeptides and compositions includingpolypeptides. As used herein, “polypeptide” refers to a polymer of aminoacids linked by peptide bonds. Thus, for example, the terms peptide,oligopeptide, protein, and enzyme are included within the definition ofpolypeptide. This term also includes post-expression modifications ofthe polypeptide, for example, glycosylations, acetylations,phosphorylations and the like. The term polypeptide does not connote aspecific length of a polymer of amino acids. A polypeptide may beobtainable directly from a natural source, or can be prepared with theaid of recombinant, enzymatic, or chemical techniques. In the case of apolypeptide or polynucleotide that is naturally occurring, suchpolypeptide or polynucleotide is typically isolated. An “isolated”polypeptide is one that has been removed from its natural environment.For instance, an “isolated” polypeptide is a polypeptide that has beenremoved from the cytoplasm or from the outer membrane of a cell, andmany of the polypeptides, nucleic acids, and other cellular material ofits natural environment are no longer present. A “purified” polypeptideis one that is at least 60% free, preferably 75% free, and mostpreferably 90% free from other components with which they are naturallyassociated. Polypeptides that are produced outside the organism in whichthey naturally occur, e.g., through chemical or recombinant means, areconsidered to be isolated and purified by definition, since they werenever present in a natural environment. Unless otherwise specified, “a,”“an,” “the,” and “at least one” are used interchangeably and mean one ormore than one.

The polypeptides of the present invention are obtainable from a memberof the family Campylobacteriaceae, (Vandamme et al. Int. J. Syst.Bacteriol. 41: 451-455 (1991)), preferably the genus Campylobacter. Amember of the genus Campylobacter is also referred to herein asCampylobacter spp. Examples of Campylobacter spp. from whichpolypeptides of the present invention may be obtained include C.hyointestinalis, C. mucosalis, C. concisus, C. sputorum, C. jejuni, C.coli, C. lari, C. upsaliensis, C. rectus, C. curvus, C. hominis, C.fetus, C, intestinalis and C. doylei. Preferably, the Campylobacter spp.from which polypeptides of the present invention may be obtained is C.jejuni. These microbes are commercially available from a depository suchas American Type Culture Collection (ATCC). In addition, such microbesare readily obtainable by isolation techniques known and used in theart. For instance, a microbe may be derived from an infected animal as afield isolate, and used to obtain polypeptides of the present inventionas described herein, or stored for future use, for example, in a frozenrepository at about −20° C. to about −95° C., in an appropriatebacteriological media containing 20% glycerol, and other like media.Methods for obtaining the polypeptides from Campylobacter spp. aredescribed herein.

A polypeptide of the present invention may be characterized by molecularweight. The molecular weight of a polypeptide, typically expressed inkilodaltons (kDa), can be determined using routine methods including,for instance, gel filtration, gel electrophoresis including sodiumdodecyl sulfate (SDS) polyacrylamide gel electrophoresis, capillaryelectrophoresis, mass spectrometry, and liquid chromatography includingHPLC.

In one aspect, the polypeptides of the present invention are metalregulated polypeptides. As used herein, a “metal regulated polypeptide”is a polypeptide that is expressed by a member of the genusCampylobacter at a greater level when the microbe is grown in low metalconditions compared to growth of same the microbe in high metalconditions. Metals are those present in the periodic table under Groups1 through 17 (IUPAC notation; also referred to as Groups I-A, II-A,IV-B, V-B, VI-B, VII-B, VIII, I-B, II-B, III-A, IV-A, V-A, VI-A, andVII-A, respectively, under CAS notation). Preferably, metals are thosein Groups 2 through 12, more preferably, Groups 3-12. Even morepreferably, the metal is iron, zinc, copper, magnesium, nickel, cobalt,manganese, molybdenum, or selenium, most preferably, iron.

For instance, one type of metal regulated polypeptide produced byCampylobacter spp. is not expressed at detectable levels during growthof the microbe in high metal conditions but is expressed at detectablelevels during growth in low metal conditions. Low metal conditions andhigh metal conditions are described in greater detail herein. Examplesof such metal regulated polypeptides obtainable from a Campylobacterspp. have molecular weights (as determined by separation of thepolypeptides using a stacking gel of about 4% on a resolving gel ofabout 10% under reducing and denaturing conditions of between 150 kDaand 152 kDa, between 143 kDa and 145 kDa, between 123 kDa and 125 kDa,between 92 kDa and 94 kDa, between 88 kDa and 90 kDa, between 73 kDa and75 kDa, between 69 kDa and 71 kDa, between 50 kDa and 53 kDa, or between38 kDa and 40 kDa. Preferably, the metal regulated polypeptides havemolecular weights of 151 kDa, 144 kDa, 124 kDa, 93 kDa, 89 kDa, 74 kDa,70 kDa, 52 kDa, 51 kDa, or 39 kDa.

Another type of metal regulated polypeptide produced by Campylobacterspp. is expressed at detectable levels during growth of the microbe inhigh metal conditions but expressed at higher levels during growth inlow metal conditions. The expression of such polypeptides is referred toherein as “enhanced” during growth in low metal conditions. Examples ofmetal regulated polypeptides showing enhanced expression and obtainablefrom Campylobacter spp. have molecular weights (as determined byseparation of the polypeptides using an about 10% SDS-PAGE gel underreducing and denaturing conditions) of between 57 kDa and 59 kDa,between 54 kDa and 56 kDa, between 47 kDa and 49 kDa, between 42 kDa and44 kDa, between 37 kDa and 39 kDa, or between 28 kDa and 30 kDa.Preferably, the metal regulated polypeptides having enhanced expressionhave molecular weights of 58 kDa, 55 kDa, 48 kDa, 43 kDa, 38 kDa, or 29kDa.

Whether a metal regulated polypeptide is expressed at a detectable levelor has enhanced expression during growth in low metal conditions can bedetermined by methods useful for comparing the presence of polypeptides,including, for example, gel filtration, gel electrophoresis includingsodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis,capillary electrophoresis, mass spectrometry, and liquid chromatographyincluding HPLC. Separate cultures of a Campylobacter spp. are grownunder high metal conditions and under low metal conditions, polypeptidesof the present invention are isolated as described herein, and thepolypeptides present in each culture are resolved and compared.Typically, an equal amount of polypeptide from each culture is used. Forinstance, when SDS polyacrylamide gel electrophoresis is used to comparethe polypeptides, about 30 μg micrograms of polypeptide from eachculture is used and loaded into a well. After running the gel andstaining the polypeptides, the two lanes can be compared.

Preferably, polypeptides of the present invention have immunogenicactivity. “Immunogenic activity” refers to the ability of a polypeptideto elicit an immunological response in an animal. An immunologicalresponse to a polypeptide is the development in an animal of a cellularand/or antibody-mediated immune response to the polypeptide. Usually, animmunological response includes but is not limited to one or more of thefollowing effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells, directed to anepitope or epitopes of the polypeptide. “Epitope” refers to the site onan antigen to which specific B cells and/or T cells respond so thatantibody and/or a cellular immune response are produced.

Also provided by the present invention are whole cell preparations of amicrobe, where the microbe expresses one or more of the polypeptides ofthe present invention. The cells present in a whole cell preparation arepreferably inactivated such that the cells cannot replicate, but theimmunogenicity of the polypeptides of the present invention expressed bythe microbe is maintained. Typically, the cells are killed by exposureto agents such as glutaraldehyde, formalin, or formaldehyde.

Compositions

The present invention also provides compositions including at leastabout 1 of the polypeptides of the present invention, more preferably atleast about 2, at least about 3, at least about 4, and so on, to about 8polypeptides of the present invention. A composition can includepolypeptides obtainable from 1 species of Campylobacter, or can beobtainable from a combination of 2 or more species of Campylobacter, forinstance, C. jejuni and a second Campylobacter other than C. jejuni.Furthermore, a composition can include polypeptides obtainable from 2 ormore strains of the same species of Campylobacter. For instance, acomposition can include polypeptides obtainable from 2 differentisolates of C. jejuni.

Optionally, a polypeptide of the present invention can be covalentlybound to a carrier polypeptide to improve the immunological propertiesof the polypeptide. Useful carrier polypeptides are known to the art.The chemical coupling of a polypeptide of the present invention can becarried out using known and routine methods. For instance, varioushomobifunctional and/or heterobifunctional cross-linker reagents such asbis(sulfosuccinimidyl)suberate, bis(diazobenzidine), dimethyladipimidate, dimethyl pimelimidate, dimethyl superimidate,disuccinimidyl suberate, glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide,sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide, sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfosuccinimidyl4-(p-maleimido-phenyl) butyrate and(1-ethyl-3-(dimethyl-aminopropyl)carbodiimide can be used (Harlow andLane, Antibodies, A Laboratory Manual, generally and Chapter 5, ColdSpring Harbor Laboratory, Cold Spring Harbor, New York, N.Y. (1988)).

Preferably, such compositions of the present invention include lowconcentrations of lipopolysaccharide (LPS). LPS is a component of theouter membrane of most gram negative microbes (see, for instance,Nikaido and Vaara, Outer Membrane, In: Escherichia coli and Salmonellatyphimurium, Cellular and Molecular Biology, Neidhardt et al., (eds.)American Society for Microbiology, Washington, D.C., pp. 7-22 (1987),and typically includes polysaccharides (O-specific chain, the outer andinner core) and the lipid A region. The lipid A component of LPS is themost biologically active component of the LPS structure and togetherinduce a wide spectrum of pathophysiological effects in mammals. Themost dramatic effects are fever, disseminated intravascular coagulation,complement activation, hypotensive shock, and death. The non-specificimmunostimulatory activity of LPS can enhance the formation of agranuloma at the site of administration of compositions that includeLPS. Such reactions can result in undue stress on the animal by whichthe animal may back off feed or water for a period of time, andexasperate infectious conditions in the animal. In addition, theformation of a granuloma at the site of injection can increase thelikelihood of possible down grading of the carcass due to scaring orblemishes of the tissue at the injection site (see, for instance, Rae,Injection Site Reactions, available atwww.animal.ufl.edu/short94/rae.htm).

The concentration of LPS can be determined using routine methods knownto the art. Such methods typically include measurement of dye binding byLPS (see, for instance, Keler and Nowotny, Analyt. Biochem., 156, 189(1986)) or the use of a Limulus amebocyte lysate (LAL) test (see, forinstance, Endotoxins and Their Detection With the Limulus AmebocyteLystate Test, Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y.(1982)). There are four basic commercially available methods that aretypically used with an LAL test: the gel-clot test; the turbidimetric(spectrophotometric) test; the colorimetric test; and the chromogenictest. An example of a gel-clot assay is available under the tradenameE-TOXATE (Sigma Chemical Co., St. Louis, Mo.; see Sigma TechnicalBulletin No. 210), and PYROTELL (Associates of Cape Cod, Inc., EastFalmouth, Mass.). Typically, assay conditions include contacting thecomposition with a preparation containing a lysate of the circulatingamebocytes of the horseshoe crab, Limulus polyphemus. When exposed toLPS, the lysate increases in opacity as well as viscosity and may gel.About 0.1 milliliter of the composition is added to lysate. Typically,the pH of the composition is between 6 and 8, preferably, between 6.8and 7.5. The mixture of composition and lysate is incubated for about 1hour undisturbed at about 37° C. After incubation, the mixture isobserved to determine if there was gelation of the mixture. Gelationindicates the presence of endotoxin. To determine the amount ofendotoxin present in the composition, dilutions of a standardizedsolution of endotoxin are made and tested at the same time that thecomposition is tested. Standardized solutions of endotoxin arecommercially available from, for instance, Sigma Chemical (Catalog No.210-SE), U.S. Pharmacopeia (Rockville, Md., Catalog No. 235503), andAssociates of Cape Cod, Inc., (Catalog No. E0005). In general, when acomposition of the present invention is prepared by isolatingpolypeptides from a microbe by a method as described herein (e.g., amethod that includes disrupting and solubilizing the cells, andcollecting the insoluble polypeptides), the amount of LPS in acomposition of the present invention is less than the amount of LPSpresent in a mixture of the same amount of the microbe that has beendisrupted under the same conditions but not solubilized. Typically, thelevel of LPS in a composition of the present invention is decreased by,in increasing order of preference, at least about 50%, at least about60%, at least about 70%, at least about 80%, or at least about 90%relative to the level of LPS in a composition prepared by disrupting,but not solubilizing, the same microbe.

The present invention also provides compositions including a whole cellpreparation of at least 1, at least 2, at least 3, at least 4, at least5, or 6 Campylobacter spp.

The compositions of the present invention optionally further include apharmaceutically acceptable carrier. “Pharmaceutically acceptable”refers to a diluent, carrier, excipient, salt, etc, that is compatiblewith the other ingredients of the composition, and not deleterious tothe recipient thereof. Typically, the composition includes apharmaceutically acceptable carrier when the composition is used asdescribed herein. The compositions of the present invention may beformulated in pharmaceutical preparations in a variety of forms adaptedto the chosen route of administration, including routes suitable forstimulating an immune response to an antigen. Thus, a composition of thepresent invention can be administered via known routes including, forexample, oral; parental including intradermal, subcutaneous,intramuscular, intravenous, intraperitoneal, etc., and topically, suchas, intranasal, intrapulmonary, intramammary, intravaginal,intrauterine, intradermal, and rectally etc. It is foreseen that acomposition can be administered to a mucosal surface, such as byadministration to the nasal or respiratory mucosa (e.g. spray oraerosol), to stimulate mucosal immunity, such as production of secretoryIgA antibodies, throughout the animal's body.

A composition of the present invention can also be administered via asustained or delayed release implant. Implants suitable for useaccording to the invention are known and include, for example, thosedisclosed in Emery and Straub (WO 01/37810 (2001)), and Emery et al. (WO96/01620 (1996)). Implants can be produced at sizes small enough to beadministered by aerosol or spray. Implants also include nanospheres andmicrospheres.

A composition of the present invention is administered in an amountsufficient to treat certain conditions as described herein. The amountof polypeptides or whole cells present in a composition of the presentinvention can vary. For instance, the dosage of polypeptides can bebetween 0.01 micrograms (μg) and 300 mg, typically between 0.1 mg and 10mg. When the composition is a whole cell preparation, the cells can bepresent at a concentration of, for instance, 10⁶ bacteria/ml, 10⁷bacteria/ml, 10⁸ bacteria/ml, or 10⁹ bacteria/ml. For an injectablecomposition (e.g. subcutaneous, intramuscular, etc.) the polypeptidesmay be present in the composition in an amount such that the totalvolume of the composition administered is 0.5 ml to 5.0 ml, typically1.0-2.0 ml. When the composition is a whole cell preparation, the cellsare preferably present in the composition in an amount that the totalvolume of the composition administered is 0.5 ml to 5.0 ml, typically1.0-2.0 ml. The amount administered will vary depending on variousfactors including, but not limited to, the specific polypeptides chosen,the weight, physical condition and age of the animal, and the route ofadministration. Thus, the absolute weight of the polypeptide included ina given unit dosage form can vary widely, and depends upon factors suchas the species, age, weight and physical condition of the animal, aswell as the method of administration. Such factors can be determined byone of skill in the art. Other examples of dosages suitable for theinvention are disclosed in Emery et al. (U.S. Pat. No. 6,027,736).

The formulations may be conveniently presented in unit dosage form andmay be prepared by methods well known in the art of pharmacy. Allmethods of preparing a composition including a pharmaceuticallyacceptable carrier include the step of bringing the active compound(e.g., a polypeptide or whole cell of the present invention) intoassociation with a carrier that constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into the desired formulations.

A composition including a pharmaceutically acceptable carrier can alsoinclude an adjuvant. An “adjuvant” refers to an agent that can act in anonspecific manner to enhance an immune response to a particularantigen, thus potentially reducing the quantity of antigen necessary inany given immunizing composition, and/or the frequency of injectionnecessary in order to generate an adequate immune response to theantigen of interest. Adjuvants may include, for example, IL-1, IL-2,emulsifiers, muramyl dipeptides, dimethyldiocradecylammonium bromide(DDA), pyridine, aluminum hydroxide, oils, saponins, alpha-tocopherol,polysaccharides, emulsified paraffins (including, for instance, thoseavailable from under the tradename EMULSIGEN from MVP Laboratories,Ralston, Nebr.), ISA-70, RIBI and other substances known in the art.

In another embodiment, a composition of the invention including apharmaceutically acceptable carrier can include a biological responsemodifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-alpha,IFN-gamma, and other cytokines that effect immune cells. An immunizingcomposition can also include other components known to the art such asan antibiotic, a preservative, an anti-oxidant, or a chelating agent.

Methods of Making

Polypeptides and whole cell preparations of the present invention may beobtained by incubating a member of the genus Campylobacter underconditions that promote expression of one or more of the polypeptidesdescribed herein. The present invention also includes compositionsprepared by the processes disclosed herein. Typically, such conditionsare low metal conditions. As used herein, the phrase “low metalconditions” refers to an environment, typically bacteriological media,that contains amounts of a free metal that cause a microbe to expressmetal regulated polypeptides. As used herein, the phrase “high metalconditions” refers to an environment that contains amounts of a freemetal that cause a microbe to either not express one or more of themetal regulated polypeptides described herein, or to decrease expressionof such a polypeptide. Low metal conditions are generally the result ofthe addition of a metal chelating compound to a bacteriological medium.High metal conditions are generally present when a chelator is notpresent in the medium, and/or a metal is added to the medium. Examplesof metal chelators include natural and synthetic compounds. Examples ofnatural compounds include plant phenolic compounds, such as flavenoids.Examples of flavenoids include the copper chelators catechin andnaringenin, and the iron chelators myricetin and quercetin. Examples ofsynthetic copper chelators include, for instance, tetrathiomolybdate,and examples of synthetic zinc chelators include, for instance,N,N,N′,N′-Tetrakis (2-pyridylmethyl)-ethylene diamine. Examples ofsynthetic iron chelators include 2,2′-dipyridyl (also referred to in theart as α,α′-bipyridyl), 8-hydroxyquinoline,ethylenediamine-di-O-hydroxyphenylacetic acid (EDDHA), desferrioxaminemethanesulphonate (desferol), transferrin, lactoferrin, ovotransferrin,biological siderophores, such as, the catecholates and hydroxamates, andcitrate. Preferably, 2,2′-dipyridyl is used for the chelation of iron.Typically, 2,2′-dipyridyl is added to the media at a concentration of atleast 0.0025 micrograms/milliliter (μg/ml), at least 0.025 μg/ml, or atleast 0.25 μg/ml. High levels of 2,2′-dipyridyl can be 10 μg/ml, 20μg/ml, or 30 μg/ml.

It is expected that a Campylobacter spp. with a mutation in a fur genewill result in the constitutive expression of many, if not all, of themetal regulated polypeptides of the present invention. A fur gene hasbeen identified in a C. jejuni (van VLiet et al., J. Bacteriol., 180,5291-5298, (1998)). The production of a fur mutation in a Campylobacterspp. can be produced using routine methods including, for instance,electroporation and genetic constructs useful for gene knock-out in gramnegative bacteria.

Many Campylobacter spp. are able to grow in low metal conditions invitro in artificial media only after adaptation. For instance, aCampylobacter spp. can be adapted to low iron conditions in vitro bygrowth in the presence of low concentrations of an iron chelator and,after growth in a medium containing the chelator, gradually increasingthe concentration of the chelator. For instance, a Campylobacter spp.can be adapted to growth in low iron conditions by adding 10 μg/ml of2,2′-dipyridyl to a medium, and gradually increasing the concentrationof the chelator to a greater concentration, for instance, 20 μg/ml.

The medium used to incubate the microbe and the volume of media used toincubate the microbe can vary. When a Campylobacter spp. microbe isbeing evaluated for the ability to produce the polypeptides describedherein, the microbe can be grown in a suitable volume, for instance, 10milliliters to 1 liter of medium. When a microbe is being grown toobtain polypeptides for use in, for instance, administration to animals,the microbe may be grown in a fermentor to allow the isolation of largeramounts of polypeptides. Methods for growing microbes in a fermentor areroutine and known to the art. The conditions used for growing a microbepreferably include a metal chelator, more preferably an iron chelator,for instance 2,2′-dipyridyl, a pH of between about 6.5 and about 7.5,preferably between about 6.9 and 7.1, and a temperature of about 37° C.When a fermentor is used, the culture may be purged with an appropriategas, for instance, carbon dioxide, to maintain microaerophilicconditions. Members of the genus Campylobacter are microaerophilicorganism, thus growth conditions do not include levels of oxygen thatwill prevent growth.

In some aspects of the invention, a Campylobacter spp. may be harvestedafter growth. Harvesting includes concentrating the microbe into asmaller volume and suspending in a media different than the growthmedia. Methods for concentrating a microbe are routine and known to theart, and include, for example, filtration and/or centrifugation.Typically, the concentrated microbe is suspended in decreasing amountsof buffer. Preferably, the final buffer includes a metal chelator,preferably, ethylenediaminetetraacetic acid (EDTA). An example of abuffer that can be used contains Tris-base (7.3 grams/liter) and EDTA(0.9 grams/liter), at a pH of 8.5. Optionally, the final buffer alsominimizes proteolytic degradation. This can be accomplished by havingthe final buffer at a pH of greater than 8.0, preferably, 8.5, and/orincluding one or more proteinase inhibitors (e.g., phenylmethanesulfonylfluoride). Optionally and preferably, the concentrated microbe is frozenat −20° C. or below until disrupted.

When the Campylobacter spp. is to be used as a whole cell preparation,the harvested cells may be processed using routine and know methods toinactivate the cells. Alternatively, when a Campylobacter spp. is to beused to prepare polypeptides of the present invention, the Campylobacterspp. may be disrupted using chemical, physical, or mechanical methodsroutine and known to the art, including, for example, french press,sonication, or homoginization. Preferably, homoginization is used. Asused herein, “disruption” refers to the breaking up of the cell.Disruption of a microbe can be measured by methods that are routine andknown to the art, including, for instance, changes in optical density.Typically, a microbe is subjected to disruption until the percenttransmittance is increased by 20% when a 1:100 dilution is measured. Thetemperature during disruption is typically kept low, preferably at 4°C., to further minimize proteolytic degradation.

The disrupted microbe is solubilized in a detergent, for instance, ananionic, zwitterionic, nonionic, or cationic detergent. Preferably, thedetergent is sarcosine, more preferably, sodium lauroyl sarcosinate. Asused herein, the term “solubilize” refers to dissolving cellularmaterials (e.g., polypeptides, nucleic acids, carbohydrates) into theaqueous phase of the buffer in which the microbe was disrupted, and theformation of aggregates of insoluble cellular materials. The conditionsfor solubilization preferably result in the aggregation of polypeptidesof the present invention into insoluble aggregates that are large enoughto allow easy isolation by, for instance, centrifugation.

Preferably, the sarcosine is added such that the final ratio ofsarcosine to gram weight of disrupted microbe is between 1.0 gramsarcosine per 4.5 grams pellet mass and 6.0 grams sarcosine per 4.5grams pellet mass, preferably, 4.5 gram sarcosine per 4.5 grams pelletmass. The solubilization of the microbe may be measured by methods thatare routine and known to the art, including, for instance, changes inoptical density. Typically, the solubilization is allowed to occur forat least 24 hours, more preferably, at least 48 hours, most preferably,at least 60 hours. The temperature during disruption is typically keptlow, preferably at 4° C.

The insoluble aggregates that include the polypeptides of the presentinvention may be isolated by methods that are routine and known to theart. Preferably, the insoluble aggregates are isolated bycentrifugation. Typically, centrifugation of outer membrane polypeptidesthat are insoluble in detergents requires centrifugal forces of at least50,000×g, typically 100,000×g. The use of such centrifugal forcesrequires the use of ultracentrifuges, and scale-up to process largevolumes of sample is often difficult and not economical with these typesof centrifuges. The methods described herein provide for the productionof insoluble aggregates large enough to allow the use of significantlylower centrifugal forces (for instance, 46,000×g). Methods forprocessing large volumes at these lower centrifugal forces are availableand known to the art. Thus, the insoluble aggregates can be isolated ata significantly lower cost.

Optionally and preferably, the sarcosine is removed from the isolatedpolypeptides. Methods for removing sarcosine from the isolatedpolypeptides are known to the art, and include, for instance,diafiltration, precipitation, hydrophobic chromatography, ion-exchangechromatography, and/or affinity chromatography, and ultra filtration andwashing the polypeptides in alcohol by diafiltration. After isolation,the polypeptides suspended in buffer and stored at low temperature, forinstance, −20° C. or below.

Polypeptides of the present invention may also be isolated fromCampylobacter spp. using methods that are known to the art. Theisolation of the polypeptides may be accomplished as described in, forinstance, Emery et al., (U.S. Pat. No. 5,830,479) and Emery et al.,(U.S. Patent Application US 20030036639 A1).

In those aspects of the present invention where a whole cell preparationis to be made, after growth of a Campylobacter spp. the microbe can bekilled with the addition of an agent such as glutaraldehyde, formalin,or formaldehyde, at a concentration sufficient to inactivate the cellsin the culture. For instance, formalin can be added at a concentrationof about 3% (vol:vol). After a period of time sufficient to inactivatethe cells, the cells can be harvested by, for instance, diafiltrationand/or centrifugation, and washed.

Methods of Use

An aspect of the present invention is further directed to methods ofusing the compositions of the present invention. The methods includeadministering to an animal an effective amount of a composition of thepresent invention. Preferably, the composition further includes apharmaceutically acceptable carrier. The composition can be administeredat a time that maternal antibody may be present, for instance, as earlyas one day of age, or at a later time during the life of the animal. Theanimal can be, for instance, an ungulate, a bird, a human, or acompanion animal. Examples of birds include commercial poultry such asturkeys, chickens, ducks, pheasant, and ostrich. Examples of ungulatesinclude animals that are bovine (including, for instance, cattle),caprine (including, for instance, goats), ovine (including, forinstance, sheep), porcine (including, for instance, swine), equine(including, for instance, horses), members of the family Cervidae(including, for instance, deer, elk, moose, caribou and reindeer), andBison (including, for instance, buffalo). Examples of companion animalsinclude dogs and cats.

In some aspects, the methods may further include additionaladministrations (e.g., one or more booster administrations) of thecomposition to the animal to enhance or stimulate a secondary immuneresponse. A booster can be administered at a time after the firstadministration, for instance, 1 to 8 weeks, preferably 2 to 4 weeks,after the first administration of the composition. Subsequent boosterscan be administered one, two, three, four, or more times annually.Without intending to be limited by theory, it is expected that annualboosters will not be necessary, as an animal will be challenged in thefield by exposure to members of the genus Campylobacter expressingpolypeptides having epitopes that are identical to or structurallyrelated to epitopes present on the polypeptides present in thecomposition administered to the animal.

In one aspect, the invention is directed to methods for inducing theproduction of antibody in an animal or by recombinant techniques. Theantibody produced includes antibody that specifically binds at least onepolypeptide present in the composition. In this aspect of the invention,an “effective amount” is an amount effective to result in the productionof antibody in the animal. Methods for determining whether an animal hasproduced antibodies that specifically bind polypeptides present in acomposition of the present invention can be determined as describedherein.

The method may be used to produce antibody that specifically bindspolypeptides expressed by a microbe other than the microbe from whichthe polypeptides of the composition were isolated. As used herein, anantibody that can “specifically bind” a polypeptide is an antibody thatinteracts with the epitope of the antigen that induced the synthesis ofthe antibody, or interacts with a structurally related epitope. At leastsome of the polypeptides present in the compositions of the presentinvention typically include epitopes that are conserved in thepolypeptides of different species and different genera of microbes.Accordingly, antibody produced using a composition derived from onemicrobe is expected to bind to polypeptides expressed by other microbesand provide broad spectrum protection against gram negative organisms.Examples of gram negative microbes to which the antibody specificallybinds are enteropathogens, for instance, members of the familyEnterobacteriaceae.

In one aspect the invention is also directed to treating an infection inan animal caused by a member of the genus Campylobacter. The methodincludes administering an effective amount of the composition of thepresent invention to an animal having an infection caused by a member ofthe genus Campylobacter, and determining whether the Campylobacter spp.causing the infection has decreased. Methods for deter mining whether aninfection is caused by a member of the genus Campylobacter are routineand known to the art.

In another aspect, the present invention is directed to methods fortreating one or more symptoms of certain conditions in animals,preferably humans, that may be caused by, or associated with, infectionby a member of the genus Campylobacter. Examples of conditions caused byCampylobacter spp. infections include diarrhea, fever, and abdominalcramping, as well as symptoms such as bacteremia, septic arthritis,Guilain-Barre syndrome Reiter syndrome (Peterson et al. Wes. J. Med.161: 148-152 (1994) and Allos et al. J. Infest Dis. 176: S125-128(1997)). Treatment of these conditions can be prophylactic or,alternatively, can be initiated after the development of a conditiondescribed herein. Treatment that is prophylactic, for instance,initiated before a subject manifests symptoms of a condition caused byCampylobacter spp., is referred to herein as treatment of a subject thatis “at risk” of developing the condition. Typically, an animal “at risk”of developing a condition is an animal likely to be exposed to aCampylobacter spp. causing the condition. For instance, the animal ispresent in an area where the condition has been diagnosed in at leastone other animal, or is being transported to an area where aCampylobacter spp. is endemic, and/or where conditions caused byCampylobacter spp. are prevalent. Accordingly, administration of acomposition can be performed before, during, or after the occurrence ofthe conditions described herein. Treatment initiated after thedevelopment of a condition may result in decreasing the severity of thesymptoms of one of the conditions, or completely removing the symptoms.In this aspect of the invention, an “effective amount” is an amounteffective to prevent the manifestation of symptoms of a condition,decrease the severity of the symptoms of a condition, and/or completelyremove the symptoms. The potency of a composition of the presentinvention can be tested according to routine methods (see, for instance,Stanfield et al., Microb Pathog., 3:155-165 (1987), Fox et al., Am. J.Vet. Res., 48:85-90 (1987), Ruiz-Palacios, Infect. Immun., 34:250-255(1981), and Humphrey et al., J. Infect. Dis., 151:485-493 (1985)).Methods for determining whether an animal has the conditions disclosedherein and symptoms associated with the conditions are routine and knownto the art.

The present invention is also directed to decreasing colonization of theintestinal tract or reproductive tract of an animal by a Campylobacterspp. The method includes administering an effective amount of acomposition of the present invention to an animal colonized by, or atrisk of being colonized by a gram negative microbe, preferably, aCampylobacter spp. In this aspect of the invention, an “effectiveamount” is an amount effective to decrease colonization of the animal bythe microbe. Colonization of an animal's intestinal tract by a microbecan be determined by measuring the presence of the microbe in theanimal's feces. Methods for evaluating the colonization of an animal'sreproductive tract by a microbe are routine and known to the art. It isexpected that decreasing the colonization of an animal by aCampylobacter spp. will reduce transmission of the Campylobacter spp. tohumans.

A composition of the invention can be used to provide for passiveimmunization against infection by Campylobacter spp. For instance, thecomposition can be administered to an animal to induce the production ofimmune products, such as antibodies, which can be collected from theproducing animal and administered to another animal to provide passiveimmunity. Immune components, such as antibodies, can be collected toprepare antibody compositions from serum, plasma, blood, colostrum, etc.for passive immunization therapies. Antibody compositions includingmonoclonal antibodies, anti-idiotypes, and/or recombinant antibodies canalso be prepared using known methods. Passive antibody compositions andfragments thereof, e.g., scFv, Fab, F(ab′)₂ or Fv or other modifiedforms thereof, may be administered to a recipient in the form of serum,plasma, blood, colostrum, and the like. However, the antibodies may alsobe isolated from serum, plasma, blood, colostrum, and the like, usingknown methods and spray dried or lyophilized for later use in aconcentrated or reconstituted form. Passive immunizing preparations maybe particularly advantageous for treatment of acute systemic illness, orpassive immunization of young animals that failed to receive adequatelevels of passive immunity through maternal colostrum.

Another aspect of the present invention provides methods for detectingantibody that specifically binds polypeptides of the present invention.These methods are useful in, for instance, detecting whether an animalhas antibody that specifically binds polypeptides of the presentinvention, and diagnosing whether an animal may have an infection causedby Campylobacter spp. Preferably, such diagnostic systems are in kitform. The methods include contacting an antibody with a preparation thatincludes at least one polypeptide of the present invention to result ina mixture. Preferably, the antibody is present in a biological sample,more preferably blood, serum, milk, mucosal secretions, or colostrum.The method further includes incubating the mixture under conditions toallow the antibody to specifically bind a polypeptide to form apolypeptide:antibody complex. As used herein, the term“polypeptide:antibody complex” refers to the complex that results whenan antibody specifically binds to a polypeptide. The preparation thatincludes the polypeptides present in a composition of the presentinvention may also include reagents, for instance a buffer, that provideconditions appropriate for the formation of the polypeptide:antibodycomplex. The polypeptide:antibody complex is then detected. Thedetection of antibodies is known in the art and can include, forinstance, immunofluorescence and peroxidase. The methods for detectingthe presence of antibodies that specifically bind to polypeptides of thepresent invention can be used in various formats that have been used todetect antibody, including radioimmunoassay and enzyme-linkedimmunosorbent assay.

The present invention also provides a kit for detecting antibody thatspecifically binds polypeptides of the present invention. The kitincludes at least one polypeptide of the present invention in a suitablepackaging material in an amount sufficient for at least one assay.Optionally, other reagents such as buffers and solutions needed topractice the invention are also included. Instructions for use of thepackaged polypeptides are also typically included.

As used herein, the phrase “packaging material” refers to one or morephysical structures used to house the contents of the kit. The packagingmaterial is constructed by known methods, preferably to provide asterile, contaminant-free environment. The packaging material has alabel which indicates that the polypeptides can be used for detectingantibodies induced by infection with Campylobacter spp. In addition, thepackaging material contains instructions indicating how the materialswithin the kit are employed to detect such antibodies. As used herein,the term “package” refers to a solid matrix or material such as glass,plastic, paper, foil, and the like, capable of holding within fixedlimits the polypeptides. Thus, for example, a package can be amicrotiter plate well to which microgram quantities of polypeptides havebeen affixed. “Instructions for use” typically include a tangibleexpression describing the reagent concentration or at least one assaymethod parameter, such as the relative amounts of reagent and sample tobe admixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions, and the like.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example Example 1 Production and Isolation of Metal Regulated Proteins

Campylobacter spp. jejuni can be grown under controlled fermentationconditions so as to express proteins, including proteins associated withthe outer membrane. The bacteria can be harvested and the proteins canthen be isolated and used as immunogens in a composition described indetail in the following example. Microaerophilic conditions for growthof C. jejuni on plates and in small liquid cultures were established byincubation in an anaerobic jar containing a Campy-Pak (BBL, Sparks, Md.)gas generator system. A master seed stock of a Campylobacter jejunioriginating from a turkey was prepared by inoculating the isolate into200 ml of Porcine Brain Heart Infusion Broth (P-BHI, Difco) containing0.025% metabisulfite (Sigma) and containing 10 to 20 micrograms permilliliter (μg/ml) of 2,2-dipyridyl (Sigma-Aldrich St. Louis, Mo.). Theculture was grown without stirring at 16 hours at 37° C. undermicroaerophilicc conditions. Prior to growth in a starter culture, theC. jejuni was adapted to grow in the iron chelator 2,2-dipyridyl byrepeatedly sub-culturing the isolate into increasing concentrations ofthe iron chelator, beginning at 10 μg/ml, and increasing to 20 μg/ml.The bacterium was collected by centrifugation at 10,000×g. The bacterialpellet was resuspended into 20 ml P-BHI containing 20% glycerol, andsterilely dispensed into 2 ml cryogenic vials (1 ml per vial) and storedat −90° C. The isolate was given the identification Campy-1, andestablished as a master seed. The master seed was expanded into aworking seed that was then used for the production of metal regulatedproteins. This strain was deposited with the American Type CultureCollection, P.O. Box 1549, Manassas, Va., 20108, USA, on Sep. 20, 2004.The deposit was made under the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure.

Example 2 Production of Metal Regulated Proteins

Fermentation: A cryogenic vial of the working seed (1 ml at 10⁹ CFU/ml)was used to inoculate 130 ml of 37° C. P-BHI or T-Soy containing 15-20micrograms (μg) 2,2-dipyridyl and 0.025% metabisulfite (Sigma) andincubated in an anaerobic jar containing a Campy-Pak (BBL, Sparks, Md.)gas generator system. The culture was incubated at 37° C. for 12-24hours at which point was sterilely transferred into 1.3 liters of theabove media. This second culture was allowed to grow for an additional10 hours at 37° C. This culture was used to inoculate a 20-liter BiofloIV bench-top fermentor, (New Brunswick Scientific Co, Edison N.J.)charged with 13 liters of the above-described media. The pH was heldconstant between 6.9 and 7.1 by automatic titration with 30% NaOH and10% HCL. The stirring speed was adjusted to 100 revolutions per minute(rev/minute), and the culture was maintained under microaerophilicconditions. The culture was allowed to grow continuously at theseconditions for 24 hours at which point the fermentation was terminatedby lowing the temperature of the fermentor to 10° C.

Harvest: The bacterial fermentation was concentrated and washed using aMillipore Pellicon Tangential Flow Filter assembly (MilliporeCorporation, Bedford, Mass.), equipped with a 25 ft² screen-channelseries Alpha 300K Centrasette filter (Pall Filtron). The originalculture volume of 13 liters was reduced to 2.5 liters. The bacterialretentate was then adjusted to 25 liters using physiological saline(0.85%) and then concentrated again to 2.5 liters to help remove anycontaminates not associated with the cells, e.g., secreted proteins. Theretentate (2.5 liters) was adjusted to 15 liters using sterile OsmoticShock Buffer (OMS) containing 7.26 grams/liter Tris-base and 0.93grams/liter EDTA adjusted to a pH of 8.5. The retentate was mixedthoroughly and equally dispensed (3.0 liters each) into 5 sterile fourliter Nalgene containers and placed into a −20° C. freezer for storage.The pellet mass was calculated by centrifuging 30 ml samples of thefermented culture and final harvest. Briefly, pre-weighted 50 ml Nalgeneconical tubes were centrifuged at 39,000×g for 90 minutes in a BeckmanJ2-21 centrifuge using a JA-21 rotor (Beckman Instruments, Palo AltoCalif.). At the end of the run, the supernatant was poured off and thetubes were weighed again. The pellet mass was calculated for each stage.

Disruption (Homogenization): Three liters of frozen bacterial cellslurry in OMS was thawed at 4° C. (180 g pellet mass). The liquidculture suspension was aseptically transferred into a 50 liter jacketedprocess tank containing 44 liters OMS pH 8.5 containing 0.1 gramsthimerosal/liter as preservative. The bulk bacterial suspension waschilled to 4° C. with continuous mixing for 18 hours at 200 rpm at whichtime was disrupted by homogenization. Briefly, the 50 liter tankcontaining the bacterial suspension was connected to a model 12.51 HRannie Homogenizer, (APV Systems, Rosemont, Ill.). A second 50 literjacketed process tank (empty) was connected to the homogenizer such thatthe fluid in the process tank could be passed through the homogenizer,into the empty tank and back again, allowing for multiple homogenizingpasses while still maintaining a closed system. The temperature duringhomogenization was kept at 4° C. At the start of each pass, fluid wascirculated at 70 psi through the homogenizer and back to the tank oforigin, while the homogenizer pressure was adjusted to 13,500 psi. Priorto the first pass, two pre-homogenizing samples were withdrawn from thehomogenizer to establish a baseline for determining the degree ofdisruption and monitoring of pH. The degree of disruption was monitoredby transmittance (% T at 540 nm at 1:100 dilution) compared to thenon-homogenized sample. The bacterial suspension was passed three timesthrough the homogenizer to give a final percent transmittance between78-83% T at a 1:100 dilution

After homogenization, Sodium Lauroyl Sarcosinate (Hamptosyl L-30,Chem/Serv, Minneapolis, Minn.) was aseptically added to the homogenizedbacterial suspension for solubilization. The amount of Sarcosine (30%)added equaled 0.0664 times the solubilizing volume, in liters, (1.0 gramsarcosine/4.5 grams pellet mass). The process tank was removed from thehomogenizer and kept at 4° C. while stirring at 240 rpm for 60-70 hours.

Protein harvest: The proteins within the solubilized process fluid wascollected by centrifugation using T-1 Sharples, (Alfa Laval Seperations,Warminster, Pa.). Briefly, the solubilized homogenate was fed into sixSharples with a feed rate of 250 ml/minute at 17 psi at a centrifugalforce of 60,000×g. The temperature during centrifugation was kept at 4°C. The solubilized homogenate was passed 2 times across the centrifuges.The protein was collected, resuspended and dispensed in 10 litersTris-buffer pH 8.5 containing 0.3% formalin (Sigma) as preservative.

Diafiltration: The protein suspension (10 liters) was adjusted to 60liters using sterile Tris-buffer, pH 8.5. The suspension was washed anddialyzed using a Millipore Pellicon Tangential Flow Filter assembly(Millipore Corporation), equipped with a 25 ft² screen-channel seriesAlpha 10K Centrasette filter (Pall Filtron) to remove residualsarcosine. The protein solution was concentrated by filtration to atarget volume of 10 liters at which point 50 liters of Tris-buffer pH7.4 containing 5% isopropyl alcohol was slowly added to the concentratefrom a second process tank. Isopropyl alcohol is thought to cause aslight unfolding of the protein structure allowing for the removal ofbound sarcosine without compromising the immunogenicity of the proteins.Diafiltration continued until the pH stabilized to 7.4 at which point 50liters Tris-buffer pH 7.4 was slowly added by diafiltration to removeresidual alcohol. The protein suspension was then concentrated toapproximately 5 liters. The protein concentrate was equally dispensed(500 ml) into ten sterile 1 liter Nalgene containers and stored at −20°C. until use.

Example 3 Analysis of Proteins

The protein profile of the C. jejuni isolate grown in iron-repleteand/or iron-deplete media was examined by SDS-PAGE. Briefly, theorganism was grown from a frozen master seed stock by sub-culturing into25 ml of containing 0.025% metabisulfite and 15 to 20 micrograms permilliliter (μg/ml) of 2,2-dipyridyl (Sigma-Aldrich St. Louis, Mo.)and/or P-BHI with metabisulfite containing 200 uM ferric chlorideincubated for 18 hours at 37° C. while stirring at 100 rpm. At 18 hoursof incubation, 5 ml of each culture was transferred into 500 ml ofpre-incubated (37° C.) iron-deplete and/or iron-replete media. Cultureswere allowed to grow for 18 hours at 37° C. while stirring at 100 rpm.At 18 hours post incubation each culture was centrifuged at 10,000×g for20 minutes. The bacterial pellet was resuspended in a 100 ml oftris-buffered saline and centrifuged at 10,000×g for 10 minutes toremove any contaminating media proteins. The bacterial pellet from theiron-replete and iron-deplete media was resuspended in 40 ml ofTris-buffered saline pH 7.2 and disrupted by sonication. The disruptedbacterial suspension was clarified by centrifugation at 32,000×g for 12minutes. The supernatant was collected and solubilized by the additionof sodium lauroyl sarcosinate 4% vol/vol at 4° C. for 24 hours. Thedetergent-insoluble OMP-enriched fraction was collected bycentrifugation at 32,000×g for 2.5 hours at 4° C. The OMP pellet wasresuspended in 200 μl tris-buffer at pH 7.2 and stored at −90° C. Asample of each extract was resolved on a 10% SDS-PAGE gel to compare theprotein profile obtained from cells grown in iron-replete andiron-deplete media. The gel was scanned using a BioRad GS-800densitometer to compare the difference in the protein profile of C.jejuni grown under iron-replete and iron-deplete conditions. The scannedgel is shown in FIG. 3.

Example 4 Preparation of the Immunizing Compositions Derived from C.jejuni

The composition made from C. jejuni as described in example 2 was usedto prepare a vaccine. A stock vaccine was prepared from the compositionby diluting the antigen into phosphate buffered saline (PBS) containing8.0 g/l NaCl, 0.2 g/l KCl, 1.44 g/l Na₂HPO₄ and 0.24 g/l KH₂PO₄ pH 7.4containing 10% aluminum hydroxide (Rehydrogel, Reheis Chemical CompanyBerkeley Heights, N.J.). The aluminum hydroxide suspension (500 μg totalprotein/ml) was then emulsified into the commercial adjuvant, EMULSIGEN,(MVP Laboratories, Ralston, Nebr.) using a IKA Ultra Turrax T-50homogenizing vessel (IKA, Cincinnati, Ohio). A mouse dose wasadministered to give a final dose of 50 μg total protein in a 0.1 mlinjectable volume with an adjuvant concentration of 22.5% vol/vol. Aplacebo was prepared by replacing the antigen with physiological salinein the above formulation and emulsifying the suspension into EMULSIGENto give an adjuvant concentration of 22.5%.

Example 5 Preparation of Challenge Organism

The C. jejuni isolate as described above was used for challenge.Briefly, the isolate from a frozen stock (example 1) was streaked onto ablood agar plate and incubated at 37° C. for 18 hours. Several colonieswere sub-cultured into 50 ml P-BHI containing 15 μg/ml 2,2′ dipyridyland 0.025% metabisulfite. The culture was incubated at 37° C. for 16hours, and then centrifuged at 10,000×g for 10 minutes at 4° C. topellet the bacteria. The bacterial pellet was washed once bycentrifugation (10,000×g for 15 minutes) at 4° C. The final pellet wasresuspended in 25 ml of P-BHI without dipyridyl. Just prior tochallenge, 1 ml of the above bacterial suspension was serially dilutedten fold to enumerate the number of CFU/dose.

Example 6 Mouse Vaccination and Oral Challenge Study with Campylobacterjejuni Evaluation of Fecal Shedding

In this experiment the efficacy of the C. jejuni vaccine was carried outagainst a live oral challenge in mice. The outcome parameters used toevaluate vaccine efficacy in this experiment were 1) individual mousemortality, and 2) differences in the concentration of Campylobacterbeing shed between treatment groups after challenge. Twenty (N=20)female CF-1 mice obtained from Harlan Breeding Laboratories(Indianapolis, Ind.) weighing 16-22 grams were equally distributed intotwo groups (10 mice/group). Mice were housed in polycarbonate mousecages (Ancore Corporation, Bellmore, N.Y.). Two cages were used, one foreach treatment group. Groups were designated as placebo, non-vaccinated(Group 1) and vaccinated (Group 2). Food and water were supplied adlibitum to all mice.

Mice were vaccinated three times at 14 day intervals subcutaneously withthe placebo and/or the C. jejuni vaccines described in Example 4. Thevolume of vaccine administered was 0.1 ml/mouse. Fourteen days after thethird vaccination, mice in groups 1 and 2 were orally challenged with C.jejuni at 4.05×10⁹ colony forming units (CFU) in a volume of 0.2 cc. Thechallenge organism was prepared as described in example 5.

To enumerate the difference in fecal shedding between the control andvaccinated groups, mouse droppings were collected at 12 hours postchallenge. Droppings were collected by placing a sterile pad on thefloor of each cage 1 hour prior to collection. At each time period thepad was removed and placed into a laminar flow hood. Using a sterilelyflamed forceps, twenty individual droppings were randomly collected. Theforceps were flamed between each collection so as not tocross-contaminate samples. Individual droppings were placed into sterilesaline dilution blanks (0.9 ml), two droppings per tube, to give tentubes. Each sample was macerated using a sterile 1 ml pipette andserially diluted 10 fold. Dilutions were plated on Campylobacter Agar(Difco Laboratories, Detroit, Mich.) incubated at 37 C5% CO₂ for 72hours. The number of bacteria was enumerated for each sample and thelog₁₀ colony forming units were averaged for each treatment group ateach time period.

Table 1 shows the difference in the fecal shedding between vaccinatedand non-vaccinated mice after an oral challenge with C. jejuni at eachtime period. There was a large difference between treatment groups inthe amount of Campylobacter shedding in feces post-challenge. Thechallenge dose represented as time 0 in Table 1 shows the initialinoculum given to each mouse. Within twelve hours post challenge therewas a dramatic decrease in the amount of Campylobacter being shed fromthe vaccinated group as compared to the Placebo group. Averaged acrossthe study period and accounting for repeated estimates, vaccinates shedless Campylobacter at each sampling period when compared tonon-vaccinates, with a degree of significance of P=0.005. The amount ofCampylobacter being shed in the vaccinated group dramatically declinedwith each sampling period as compared to the non-vaccinated Placebogroup (FIG. 2). At 12 hours post challenge the difference in the amountof Campylobacter being shed between the vaccinated and non-vaccinatedgroup was greater then 3 logs (Table 1, FIG. 2).

TABLE 1 The Difference in Shedding of Campylobacter jejuni Between theNon-Vaccinated and Vaccinated Treatment Groups after Oral Challenge.Mean log₁₀ Colony Forming Units Group 1 Sampling Times (Non-vaccinated)Group 2 (Vaccinated) Challenge Dose (time 0) 9.607 9.607 12 hours 0(a)3.865 (a)Detection limit of the test was 10⁻¹

The experiment was terminated at 12 hours due to a contamination with aPseudomonas aeruginosa which grew through the selective antibiotics inthe Campylobacter agar at all subsequent samplings. No mortality wasobserved in any mice after challenge. The results clearly demonstratethat subcutaneous vaccination with the composition results in asignificant difference (P=0.005) in the colonization of Campylobactercompared to a non-vaccinated Placebo group.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. The foregoing detaileddescription and examples have been given for clarity of understandingonly. No unnecessary limitations are to be understood therefrom. Theinvention is not limited to the exact details shown and described, forvariations obvious to one skilled in the art will be included within theinvention defined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1. An isolated metal regulated polypeptide obtainable from aCampylobacter spp., wherein the polypeptide is expressed by aCampylobacter spp. at a detectable level during growth under low metalconditions and is not expressed by the Campylobacter spp. at adetectable level during growth in high metal conditions. 2.-27.(canceled)
 28. A composition comprising: Campylobacter jejuni wholecells comprising iron-regulated polypeptides having molecular weights,as determined by 10% SDS-PAGE, of between 150 kDa and 152 kDa, between143 kDa and 145 kDa, between 123 kDa and 125 kDa, between 88 kDa and 90kDa, between 73 kDa and 75 kDa, between 69 kDa and 71 kDa, between 51kDa and 53 kDa, between 50 kDa and 52 kDa, or between 38 kDa and 40 kDa;and a pharmaceutically acceptable carrier.
 29. A method comprising:administering to an animal colonized by or at risk of being colonized byCampylobacter jejuni an amount of a composition effective to decreasecolonization of the intestinal tract of the animal by C. jejuni, whereinthe composition comprises whole C. jejuni cells that compriseiron-regulated polypeptides having molecular weights, as determined by10% SDS-PAGE, of between 150 kDa and 152 kDa, between 143 kDa and 145kDa, between 123 kDa and 125 kDa, between 88 kDa and 90 kDa, between 73kDa and 75 kDa, between 69 kDa and 71 kDa, between 51 kDa and 53 kDa,between 50 kDa and 52 kDa, or between 38 kDa and 40 kDa.
 30. The methodof claim 29 wherein the animal is a mammal or a bird.
 31. The method ofclaim 29 wherein the C. jejuni is C. jejuni ATCC strain PTA-6215.
 32. Amethod of reducing fecal shedding of Campylobacter jejuni in an animalcolonized by C. jejuni, the method comprising: administering to theanimal an amount of a composition effective to decrease fecal sheddingof C. jejuni by the animal, wherein the composition comprises whole C.jejuni cells that comprise iron-regulated polypeptides having molecularweights, as determined by 10% SDS-PAGE, of between 150 kDa and 152 kDa,between 143 kDa and 145 kDa, between 123 kDa and 125 kDa, between 88 kDaand 90 kDa, between 73 kDa and 75 kDa, between 69 kDa and 71 kDa,between 51 kDa and 53 kDa, between 50 kDa and 52 kDa, or between 38 kDaand 40 kDa.
 33. The method of claim 32 wherein the animal is a mammal ora bird.
 34. The method of claim 32 wherein the C. jejuni is C. jejuniATCC strain PTA-6215.